Characterization of the basic leucine zipper transcription factor family of Neoporphyra haitanensis and its role in acclimation to dehydration stress.

BACKGROUND: Neoporphyra haitanensis, a major marine crop native to southern China, grows in the harsh intertidal habitats of rocky coasts. The thallus can tolerate fluctuating and extreme environmental stresses, for example, repeated desiccation/rehydration due to the turning tides. It is also a typical model system for investigating stress tolerance mechanisms in intertidal seaweed. The basic leucine zipper (bZIP) transcription factors play important roles in the regulation of plants' responses to environmental stress stimuli. However, little information is available regarding the bZIP family in the marine crop Nh. haitanensis. RESULTS: We identified 19 bZIP genes in the Nh. haitanensis genome and described their conserved domains. Based on phylogenetic analysis, these 19 NhhbZIP genes, distributed unevenly on the 11 superscaffolds, were divided into four groups. In each group, there were analogous exon/intron numbers and motif compositions, along with diverse exon lengths. Cross-species collinearity analysis indicated that 17 and 9 NhhbZIP genes were orthologous to bZIP genes in Neopyropia yezoensis and Porphyra umbilicalis, respectively. Evidence from RNA sequencing (RNA-seq) data showed that the majority of NhhbZIP genes (73.68%) exhibited transcript abundance in all treatments. Furthermore, genes NN 2, 4 and 5 showed significantly altered expression in response to moderate dehydration, severe dehydration, and rehydration, respectively. Gene co-expression network analysis of the representative genes was carried out, followed by gene set enrichment analysis. Two NhhbZIP genes collectively responding to dehydration and rehydration and their co-expressing genes mainly participated in DNA repair, DNA metabolic process, and regulation of helicase activity. Two specific NhhbZIP genes responding to severe dehydration and their corresponding network genes were mainly involved in macromolecule modification, cellular catabolic process, and transmembrane transport. Three specific NhhbZIP genes responding to rehydration and their co-expression gene networks were mainly involved in the regulation of the cell cycle process and defense response. CONCLUSIONS: This study provides new insights into the structural composition, evolution, and function of the NhhbZIP gene family. Our results will help us to further study the functions of bZIP genes in response to dehydration and rehydration in Nh. haitanensis and improve Nh. haitanensis in southern China.

Genome-wide analysis of HSP70 gene superfamily in Pyropia yezoensis (Bangiales, Rhodophyta): identification, characterization and expression profiles in response to dehydration stress.

BACKGROUND: Heat shock proteins (HSPs) perform a fundamental role in protecting plants against abiotic stresses. Individual family members have been analyzed in previous studies, but there has not yet been a comprehensive analysis of the HSP70 gene family in Pyropia yezoensis. RESULTS: We investigated 15 putative HSP70 genes in Py. yezoensis. These genes were classified into two sub-families, denoted as DnaK and Hsp110. In each sub-family, there was relative conservation of the gene structure and motif. Synteny-based analysis indicated that seven and three PyyHSP70 genes were orthologous to HSP70 genes in Pyropia haitanensis and Porphyra umbilicalis, respectively. Most PyyHSP70s showed up-regulated expression under different degrees of dehydration stress. PyyHSP70-1 and PyyHSP70-3 were expressed in higher degrees compared with other PyyHSP70s in dehydration treatments, and then expression degrees somewhat decreased in rehydration treatment. Subcellular localization showed PyyHSP70-1-GFP and PyyHSP70-3-GFP were in the cytoplasm and nucleus/cytoplasm, respectively. Similar expression patterns of paired orthologs in Py. yezoensis and Py. haitanensis suggest important roles for HSP70s in intertidal environmental adaptation during evolution. CONCLUSIONS: These findings provide insight into the evolution and modification of the PyyHSP70 gene family and will help to determine the functions of the HSP70 genes in Py. yezoensis growth and development.

Comparative Transcriptome Analysis Provides Insights into Response of Ulva compressa to Fluctuating Salinity Conditions.

Ulva compressa, a green tide-forming species, can adapt to hypo-salinity conditions, such as estuaries and brackish lakes. To understand the underlying molecular mechanisms of hypo-salinity stress tolerance, transcriptome-wide gene expression profiles in U. compressa were created using digital gene expression profiles. The RNA-seq data were analyzed based on the comparison of differently expressed genes involved in specific pathways under hypo-salinity and recovery conditions. The up-regulation of genes in photosynthesis and glycolysis pathways may contribute to the recovery of photosynthesis and energy metabolism, which could provide sufficient energy for the tolerance under long-term hyposaline stress. Multiple strategies, such as ion transportation and osmolytes metabolism, were performed to maintain the osmotic homeostasis. Additionally, several long noncoding RNA were differently expressed during the stress, which could play important roles in the osmotolerance. Our work will serve as an essential foundation for the understanding of the tolerance mechanism of U. compressa under the fluctuating salinity conditions.

Floridean Starch and Floridoside Metabolic Pathways of Neoporphyra haitanensis and Their Regulatory Mechanism under Continuous Darkness.

Floridean starch and floridoside are the main storage carbohydrates of red algae. However, their complete metabolic pathways and the origin, function, and regulatory mechanism of their pathway genes have not been fully elucidated. In this study, we identified their metabolic pathway genes and analyzed the changes in related gene expression and metabolite content in Neoporphyra haitanensis under continuous dark conditions. Our results showed that genes from different sources, including eukaryotic hosts, cyanobacteria, and bacteria, were combined to construct floridean starch and floridoside metabolic pathways in N. haitanensis. Moreover, compared with those in the control, under continuous dark conditions, floridean starch biosynthesis genes and some degradation genes were significantly upregulated with no significant change in floridean starch content, whereas floridoside degradation genes were significantly upregulated with a significant decrease in floridoside content. This implies that floridean starch content is maintained but floridoside is consumed in N. haitanensis under dark conditions. This study elucidates the "floridean starch-floridoside" metabolic network and its gene origins in N. haitanensis for the first time.

Comparative Quantitative Proteomics Reveals the Desiccation Stress Responses of the Intertidal Seaweed NEOPORPHYRA haitanensis.

Neoporphyra haitanensis is an economically important red seaweed that inhabits upper intertidal zones. The thallus tolerates extreme fluctuating environmental stresses (e.g., surviving more than 80% water loss during low tides). To elucidate the global molecular responses relevant to this outstanding desiccation tolerance, a quantitative proteomics analysis of N. haitanensis under different desiccation treatments as well as rehydration was performed. According to the clustering of expression patterns and the functional interpretation of the 483 significantly differentially expressed proteins, a three-stage cellular response to desiccation stress and subsequent rehydration was proposed. Stage I: at the beginning of water loss, multiple signal transduction pathways were triggered including lipid signaling, protein phosphorylation cascades, and histone acetylation controlling acetate biosynthesis to further modulate downstream hormone signaling. Protein protection by peptidyl-prolyl isomerase and ROS scavenging systems were also immediately switched on. Stage II: with the aggravation of stress, increases in antioxidant systems, the accumulation of LEA proteins, and the temporary biosynthesis of branched starch were observed. Multiple enzymes involved in redox homeostasis, including peroxiredoxin, thioredoxin, ascorbate peroxidase, superoxide dismutase, glutathione peroxidase, and glutathione reductase, were hypothesized to function in specific cellular compartments. Stage III: when the desiccated thalli had rehydrated for 30 mins, photosynthesis and carbon fixation were recovered, and antioxidant activities and protein structure protection were maintained at a high level. This work increases the understanding of the molecular responses to environmental stresses via a proteomic approach in red seaweeds and paves the way for further functional studies and genetic engineering.

Functional Characterization and Evolutionary Analysis of Glycine-Betaine Biosynthesis Pathway in Red Seaweed Pyropia yezoensis.

The red seaweed Pyropia yezoensis is an ideal research model for dissecting the molecular mechanisms underlying its robust acclimation to abiotic stresses in intertidal zones. Glycine betaine (GB) was an important osmolyte in maintaining osmotic balance and stabilizing the quaternary structure of complex proteins under abiotic stresses (drought, salinity, etc.) in plants, animals, and bacteria. However, the existence and possible functions of GB in Pyropia remain elusive. In this study, we observed the rapid accumulation of GB in desiccated Pyropia blades, identifying its essential roles in protecting Pyropia cells against severe osmotic stress. Based on the available genomic and transcriptomic information of Pyropia, we computationally identified genes encoding the three key enzymes in the GB biosynthesis pathway: phosphoethanolamine N-methyltransferase (PEAMT), choline dehydrogenase (CDH), and betaine aldehyde dehydrogenase (BADH). Pyropia had an extraordinarily expanded gene copy number of CDH (up to seven) compared to other red algae. Phylogeny analysis revealed that in addition to the one conservative CDH in red algae, the other six might have originated from early gene duplication events. In dehydration stress, multiple CDH paralogs and PEAMT genes were coordinating up-regulated and shunted metabolic flux into GB biosynthesis. An elaborate molecular mechanism might be involved in the transcriptional regulation of these genes.

HomBlocks: A multiple-alignment construction pipeline for organelle phylogenomics based on locally collinear block searching.

Organelle phylogenomic analysis requires precisely constructed multi-gene alignment matrices concatenated by pre-aligned single gene datasets. For non-bioinformaticians, it can take days to weeks to manually create high-quality multi-gene alignments comprising tens or hundreds of homologous genes. Here, we describe a new and highly efficient pipeline, HomBlocks, which uses a homologous block searching method to construct multiple sequence alignment. This approach can automatically recognize locally collinear blocks among organelle genomes and excavate phylogenetically informative regions to construct multiple sequence alignment in a few hours. In addition, HomBlocks supports organelle genomes without annotation and makes adjustment to different taxon datasets, thereby enabling the inclusion of as many common genes as possible. Topology comparison of trees built by conventional multi-gene and HomBlocks alignments implemented in different taxon categories shows that the same efficiency can be achieved by HomBlocks as when using the traditional method. The availability of Homblocks makes organelle phylogenetic analyses more accessible to non-bioinformaticians, thereby promising to lead to a better understanding of phylogenic relationships at an organelle genome level. AVAILABILITY AND IMPLEMENTATION: HomBlocks is implemented in Perl and is supported by Unix-like operative systems, including Linux and macOS. The Perl source code is freely available for download from https://github.com/fenghen360/HomBlocks.git, and documentation and tutorials are available at https://github.com/fenghen360/HomBlocks. CONTACT: yxmao@ouc.edu.cn or fenghen360@126.com.

Efficient Distributed Method for NLOS Cooperative Localization in WSNs.

The accuracy of cooperative localization can be severely degraded in non-line-of-sight (NLOS) environments. Although most existing approaches modify models to alleviate NLOS impact, computational speed does not satisfy practical applications. In this paper, we propose a distributed cooperative localization method for wireless sensor networks (WSNs) in NLOS environments. The convex model in the proposed method is based on projection relaxation. This model was designed for situations where prior information on NLOS connections is unavailable. We developed an efficient decomposed formulation for the convex counterpart, and designed a parallel distributed algorithm based on the alternating direction method of multipliers (ADMM), which significantly improves computational speed. To accelerate the convergence rate of local updates, we approached the subproblems via the proximal algorithm and analyzed its computational complexity. Numerical simulation results demonstrate that our approach is superior in processing speed and accuracy to other methods in NLOS scenarios.

Transcriptomic Insights into Innate Immunity Responding to Red Rot Disease in Red Alga Pyropia yezoensis.

Pyropia yezoensis, one of the most economically important marine algae, suffers from the biotic stress of the oomycete necrotrophic pathogen Pythium porphyrae. However, little is known about the molecular defensive mechanisms employed by Pyr. yezoensis during the infection process. In the present study, we defined three stages of red rot disease based on histopathological features and photosynthetic physiology. Transcriptomic analysis was carried out at different stages of infection to identify the genes related to the innate immune system in Pyr. yezoensis. In total, 2139 up-regulated genes and 1672 down-regulated genes were identified from all the infected groups. Pathogen receptor genes, including three lectin genes (pattern recognition receptors (PRRs)) and five genes encoding typical plant R protein domains (leucine rich repeat (LRR), nucleotide binding site (NBS), or Toll/interleukin-1 receptor (TIR)), were found to be up-regulated after infection. Several defense mechanisms that were typically regarded as PAMP-triggered immunity (PTI) in plants were induced during the infection. These included defensive and protective enzymes, heat shock proteins, secondary metabolites, cellulase, and protease inhibitors. As a part of the effector-triggered immunity (ETI), the expression of genes related to the ubiquitin-proteasome system (UPS) and hypersensitive cell death response (HR) increased significantly during the infection. The current study suggests that, similar to plants, Pyr. yezoensis possesses a conserved innate immune system that counters the invasion of necrotrophic pathogen Pyt. porphyrae. However, the innate immunity genes of Pyr. yezoensis appear to be more ancient in origin compared to those in higher plants.

Transcriptome-wide identification of optimal reference genes for expression analysis of Pyropia yezoensis responses to abiotic stress.

BACKGROUND: Pyropia yezoensis, a marine red alga, is an ideal research model for studying the mechanisms of abiotic stress tolerance in intertidal seaweed. Real-time quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method to analyze gene expression levels. To accurately quantify gene expression, selection and validation of stable reference genes is required. RESULTS: We used transcriptome profiling data from different abiotic stress treatments to identify six genes with relatively stable expression levels: MAP, ATPase, CGS1, PPK, DPE2, and FHP. These six genes and three conventional reference genes, UBC, EF1-α, and eif4A, were chosen as candidates for optimal reference gene selection. Five common statistical approaches (geNorm, ΔCt method, NormFinder, BestKeeper, and ReFinder) were used to identify the stability of each reference gene. Our results show that: MAP, UBC, and FHP are stably expressed in all analyzed conditions; CGS1 and UBC are stably expressed under conditions of dehydration stress; and MAP, UBC, and CGS1 are stably expressed under conditions of temperature stress. CONCLUSION: We have identified appropriate reference genes for RT-qPCR in P. yezoensis under different abiotic stress conditions which will facilitate studies of gene expression under these conditions.

Identification of proteins responding to pathogen-infection in the red alga Pyropia yezoensis using iTRAQ quantitative proteomics.

BACKGROUND: Pyropia yezoensis is an important marine crop which, due to its high protein content, is widely used as a seafood in China. Unfortunately, red rot disease, caused by Pythium porphyrae, seriously damages P. yezoensis farms every year in China, Japan, and Korea. Proteomic methods are often used to study the interactions between hosts and pathogens. Therefore, an iTRAQ-based proteomic analysis was used to identify pathogen-responsive proteins following the artificial infection of P. yezoensis with P. porphyrae spores. RESULTS: A total of 762 differentially expressed proteins were identified, of which 378 were up-regulated and 384 were down-regulated following infection. A large amount of these proteins were involved in disease stress, carbohydrate metabolism, cell signaling, chaperone activity, photosynthesis, and energy metabolism, as annotated in the KEGG database. Overall, the data showed that P. yezoensis resists infection by inhibiting photosynthesis, and energy and carbohydrate metabolism pathways, as supported by changes in the expression levels of related proteins. The expression data are available via ProteomeXchange with the identifier PXD009363. CONCLUSIONS: The current data provide an overall summary of the red algae responses to pathogen infection. This study improves our understanding of infection resistance in P. yezoensis, and may help in increasing the breeding of P. porphyrae-infection tolerant macroalgae.

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae.

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein-coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein-coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya).

Genome-wide expression profiles of Pyropia haitanensis in response to osmotic stress by using deep sequencing technology.

BACKGROUND: Pyropia haitanensis is an economically important marine crop grown in harsh intertidal habitats of southern China; it is also an excellent model system for studying mechanisms of stress tolerance. To understand the molecular mechanisms underlying osmotic tolerance and adaptation to intertidal environments, a comprehensive analysis of genome-wide gene expression profiles in response to dehydration and rehydration in Py. haitanensis was undertaken using digital gene expression profile (DGE) approaches combined with de novo transcriptome sequencing. RESULTS: RNA-sequencing of the pooled RNA samples from different developmental phases and stress treatments was performed, which generated a total of 47.7 million clean reads. These reads were de novo assembled into 28,536 unigenes (≥ 200 bp), of which 18,217 unigenes (63.83 %) were annotated in at least one reference database. DGE analysis was performed on four treatments (two biological replicates per treatment), which included moderate dehydration, severe dehydration, rehydration, and normal conditions. The number of raw reads per sample ranged from 12.47 to 15.79 million, with an average of 14.69 million reads per sample. After quality filtering, the number of clean reads per sample ranged from 11.83 to 15.04 million. All distinct sequencing reads were annotated using the transcriptome of Py. haitanensis as reference. A total of 1,681 unigenes showed significant differential expression between moderate dehydration and normal conditions, in which 977 genes were upregulated, and 704 genes were downregulated. Between severe dehydration and normal conditions, 1,993 unigenes showed significantly altered expression, which included both upregulated (1,219) and downregulated genes (774). In addition, 1,086 differentially expressed genes were detected between rehydration and normal conditions, of which 720 genes were upregulated and 366 unigenes were downregulated. Most gene expression patterns in response to dehydration differed from that of rehydration, except for the synthesis of unsaturated fatty acids, several transcription factor families, and molecular chaperones that have been collectively implicated in the processes of dehydration and rehydration in Py. haitanensis. CONCLUSIONS: Taken together, these data provide a global high-resolution analysis of gene expression changes during osmotic stress that could potentially serve as a key resource for understanding the biology of osmotic acclimation in intertidal red seaweed.

Comparative transcriptome profiling of Pyropia yezoensis (Ueda) M.S. Hwang & H.G. Choi in response to temperature stresses.

BACKGROUND: Pyropia yezoensis is a model organism often used to investigate the mechanisms underlying stress tolerance in intertidal zones. The digital gene expression (DGE) approach was used to characterize a genome-wide comparative analysis of differentially expressed genes (DEGs) that influence the physiological, developmental or biochemical processes in samples subjected to 4 treatments: high-temperature stress (HT), chilling stress (CS), freezing stress (FS) and normal temperature (NT). RESULTS: Equal amounts of total RNAs collected from 8 samples (two biological replicates per treatment) were sequenced using the Illumina/Solexa platform. Compared with NT, a total of 2202, 1334 and 592 differentially expressed unigenes were detected in HT, CS and FS respectively. Clustering analysis suggested P. yezoensis acclimates to low and high-temperature stress condition using different mechanisms: In heat stress, the unigenes related to replication and repair of DNA and protein processing in endoplasmic reticulum were active; however at low temperature stresses, unigenes related to carbohydrate metabolism and energy metabolism were active. Analysis of gene differential expression showed that four categories of DEGs functioning as temperature sensors were found, including heat shock proteins, H2A, histone deacetylase complex and transcription factors. Heat stress caused chloroplast genes down-regulated and unigenes encoding metacaspases up-regulated, which is an important regulator of PCD. Cold stress caused an increase in the expression of FAD to improve the proportion of polyunsaturated fatty acids. An up-regulated unigene encoding farnesyl pyrophosphate synthase was found in cold stress, indicating that the plant hormone ABA also played an important role in responding to temperature stress in P. yezoensis. CONCLUSION: The variation of amount of unigenes and different gene expression pattern under different temperature stresses indicated the complicated and diverse regulation mechanism in response to temperature stress in P. yezoensis. Several common metabolism pathways were found both in P. yezoensis and in higher plants, such as FAD in low-temperature stress and HSP in heat stress. Meanwhile, many chloroplast genes and unigene related to the synthesis of abscisic acid were detected, revealing its unique temperature-regulation mechanism in this intertidal species. This sequencing dataset and analysis may serve as a valuable resource to study the mechanisms involved in abiotic stress tolerance in intertidal seaweeds.

Chromosome-level genome assembly of humpback grouper using PacBio HiFi reads and Hi-C technologies.

The humpback grouper (Cromileptes altivelis), a medium-sized coral reef teleost, is a naturally rare species distributed in the tropical waters of the Indian and Pacific Oceans. It has high market value, but artificial reproduction and breeding remain limited and need to be improved. Here, we assembled the genome with 1.08 Gb, with a contig N50 of 43.78 Mb. A total of 96.59% of the assembly anchored to 24 pseudochromosomes using Hi-C technology. It contained 24,442 protein-coding sequences, of which 99.3% were functionally annotated. The completeness of the assembly was estimated to be 97.3% using BUSCO. The phylogenomic analysis suggested that humpback grouper should be classified into the genus Epinephelus rather than Cromileptes. The comparative genomic analysis revealed that the gene families related to circadian entrainment were significantly expanded. The high-quality reference genome provides useful genomic tools for exploiting the genomic resource of humpback grouper and supports the functional genomic study of this species in the future.

Characteristics of upper respiratory tract rhinovirus in children with allergic rhinitis and its role in disease severity.

Allergic rhinitis (AR) is a global health challenge that particularly affects the quality of life of children. Human rhinovirus (HRV) infection usually causes common cold in the upper respiratory tract (URT) and can also affect airway allergy development, such as asthma exacerbation, but its relationship with AR is poorly understood. The study aimed to gain insight into the characteristics of HRV that is prevalent in AR children and its role in AR severity. A total of 362 children with symptomatic AR were enrolled from southwestern China during 2022-2023, and nasal lavage samples were collected for HRV molecular characterization and cytokine measurement. HRV was detected in 40% of the AR children, with peak detection in autumn. The positive rate was not correlated with whether the subjects were under allergen-specific immunotherapy (AIT). Among the detected HRVs, 42% were species A, 36% were species B, and 22% were species C, involving 21 A genotypes, 6 B genotypes, and 7 C genotypes. HRV positivity was significantly associated with symptom severity (visual analog scale [VAS] score) and elevated levels of local nasal IgE, interleukin-25 (IL-25), IL-4, and CXCL13 in AR children who did not receive antiallergic treatment. All three species of HRV strains (A1B, A21, B27, B70, and C17) had been isolated and were able to infect respiratory epithelial tissue in vitro. Complete genome sequencing showed that the antigenic epitopes of the isolated HRVs had certain variations. Our work reveals the etiological characteristics of URT-HRV in AR children and suggests a role of HRV infection in the pathogenesis of childhood AR. IMPORTANCE: Our study revealed high human rhinovirus (HRV) detection rate in children with allergic rhinitis (AR), and HRV infection (A, B, or C species) is positively associated with the symptom severity in AR children. Elevated nasal IgE, interleukin-25 (IL-25), IL-4, and CXCL13 levels suggest a potential pathogenic mechanism by which HRV infection induces nasal type 2 immune/inflammation responses and local IgE production in AR patients. In addition, etiological analysis found that the main prevalent HRV species in AR children are A and B (~80%), which is different from acute respiratory infection and asthma exacerbation, where species A and C are dominant. The data reveal the distinct species prevalence characteristics of HRV infection in AR. Finally, we isolated all three species of HRV strains from nasal cavity of AR children with varying degrees of antigenic epitope mutations and in vitro infectivity, highlighting the importance of strengthening monitoring and intervention for respiratory HRV infection in AR children.

Genome-Wide Identification and Analysis of MYB Transcription Factors in Pyropia yezoensis.

MYB transcription factors are one of the largest transcription factor families in plants, and they regulate numerous biological processes. Red algae are an important taxonomic group and have important roles in economics and research. However, no comprehensive analysis of the MYB gene family in any red algae, including Pyropia yezoensis, has been conducted. To identify the MYB gene members of Py. yezoensis, and to investigate their family structural features and expression profile characteristics, a study was conducted. In this study, 3 R2R3-MYBs and 13 MYB-related members were identified in Py. yezoensis. Phylogenetic analysis indicated that most red algae MYB genes could be clustered with green plants or Glaucophyta MYB genes, inferring their ancient origins. Synteny analysis indicated that 13 and 5 PyMYB genes were orthologous to Pyropia haitanensis and Porphyra umbilicalis, respectively. Most Bangiaceae MYB genes contain several Gly-rich motifs, which may be the result of an adaptation to carbon limitations and maintenance of important regulatory functions. An expression profile analysis showed that PyMYB genes exhibited diverse expression profiles. However, the expression patterns of different members appeared to be diverse, and PyMYB5 was upregulated in response to dehydration, low temperature, and Pythium porphyrae infection. This is the first comprehensive study of the MYB gene family in Py. Yezoensis and it provides vital insights into the functional divergence of MYB genes.

Cells and Fugu Response to Capsid of BFNNV Genotype.

The nervous necrosis virus (NNV) of the BFNNV genotype is the causative agent of viral encephalopathy and retinopathy (VER) in cold water fishes. Similar to the RGNNV genotype, BFNNV is also considered a highly destructive virus. In the present study, the RNA2 of the BFNNV genotype was modified and expressed in the EPC cell line. The subcellular localization results showed that the capsid and N-terminal (1-414) were located in the nucleus, while the C-terminal (415-1014) of the capsid was located in the cytoplasm. Meanwhile, cell mortality obviously increased after expression of the capsid in EPC. EPC cells were transfected with pEGFP-CP and sampled at 12 h, 24 h and 48 h for transcriptome sequencing. There are 254, 2997 and 229 up-regulated genes and 387, 1611, and 649 down-regulated genes post-transfection, respectively. The ubiquitin-activating enzyme and ubiquitin-conjugating enzyme were up-regulated in the DEGs, indicating that cell death evoked by capsid transfection may be related to ubiquitination. The qPCR results showed that heat stock protein 70 (HSP70) is extremely up-regulated after expression of BFNNV capsid in EPC, and N-terminal is the key region to evoke the high expression. For further study, the immunoregulation of the capsid in fish pcDNA-3.1-CP was constructed and injected into the Takifugu rubripes muscle. pcDNA-3.1-CP can be detected in gills, muscle and head kidney, and lasted for more than 70 d post-injection. The transcripts of IgM and interferon inducible gene Mx were up-regulated after being immunized in different tissues, and immune factors, such as IFN-γ and C3, were also up-regulated in serum, while C4 was down-regulated one week after injection. It was suggested that pcDNA-3.1-CP can be a potential DNA vaccine in stimulating the immune system of T. rubripes; however, NNV challenge needs to be conducted in the following experiments.

Quantification of Photosynthetic Pigments in Neopyropia yezoensis Using Hyperspectral Imagery.

Phycobilisomes and chlorophyll-a (Chla) play important roles in the photosynthetic physiology of red macroalgae and serve as the primary light-harvesting antennae and reaction center for photosystem II. Neopyropia is an economically important red macroalga widely cultivated in East Asian countries. The contents and ratios of 3 main phycobiliproteins and Chla are visible traits to evaluate its commercial quality. The traditional analytical methods used for measuring these components have several limitations. Therefore, a high-throughput, nondestructive, optical method based on hyperspectral imaging technology was developed for phenotyping the pigments phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), and Chla in Neopyropia thalli in this study. The average spectra from the region of interest were collected at wavelengths ranging from 400 to 1000 nm using a hyperspectral camera. Following different preprocessing methods, 2 machine learning methods, partial least squares regression (PLSR) and support vector machine regression (SVR), were performed to establish the best prediction models for PE, PC, APC, and Chla contents. The prediction results showed that the PLSR model performed the best for PE (R Test 2 = 0.96, MAPE = 8.31%, RPD = 5.21) and the SVR model performed the best for PC (R Test 2 = 0.94, MAPE = 7.18%, RPD = 4.16) and APC (R Test 2 = 0.84, MAPE = 18.25%, RPD = 2.53). Two models (PLSR and SVR) performed almost the same for Chla (PLSR: R Test 2 = 0.92, MAPE = 12.77%, RPD = 3.61; SVR: R Test 2 = 0.93, MAPE = 13.51%, RPD =3.60). Further validation of the optimal models was performed using field-collected samples, and the result demonstrated satisfactory robustness and accuracy. The distribution of PE, PC, APC, and Chla contents within a thallus was visualized according to the optimal prediction models. The results showed that hyperspectral imaging technology was effective for fast, accurate, and noninvasive phenotyping of the PE, PC, APC, and Chla contents of Neopyropia in situ. This could benefit the efficiency of macroalgae breeding, phenomics research, and other related applications.

ChIP-Based Nuclear DNA Isolation for Genome Sequencing in Pyropia to Remove Cytosol and Bacterial DNA Contamination.

Contamination from cytosolic DNA (plastid and mitochondrion) and epiphytic bacteria is challenging the efficiency and accuracy of genome-wide analysis of nori-producing marine seaweed Pyropia yezoensis. Unlike bacteria and organellar DNA, Pyropia nuclear DNA is closely associated with histone proteins. In this study, we applied Chromatin Immunoprecipitation (ChIP) of histone H3 to isolate nuclear DNA, followed by high-throughput sequencing. More than 99.41% of ChIP-sequencing data were successfully aligned to the reference nuclear genome; this was remarkably higher than those from direct extraction and direct extraction data, in which 40.96% to 42.95% are from plastids. The proportion of data that were mapped to the bacterial database when using ChIP extraction was very low. Additionally, ChIP data can cover up to 89.00% of the nuclear genome, higher than direct extraction data at equal data size and comparable to the latter at equal sequencing depth. The uncovered regions from the three methods are mostly overlapping, suggesting that incomplete sequencing accounts for the missing data, rather than failed chromatin-antibody binding in the ChIP extraction method. This ChIP extraction method can successfully separate nuclear DNA from cytosolic DNA and bacterial DNA, thus overwhelmingly reducing the sequencing cost in a genome resequencing project and providing strictly purified reference data for genome assembly. The method's applicability to other macroalgae makes it a valuable contribution to the algal research community.